Acetates

Mycelial cell wall fractionation was performed according to the method described by Fontaine et al.[86] (link) with slight modification. Briefly, wt and Δhac1 strains were grown in a 1.2-liter fermenter in liquid Sabouraud medium. After 24 h of cultivation (linear growth phase), the mycelia were collected by filtration, washed extensively with water and disrupted in a Dyno-mill (W. A. Bachofen AG, Basel, Switzerland) cell homogenizer using 0.5-mm glass beads at 4°C. The disrupted mycelial suspension was centrifuged (3,000×g for 10 min), and the cell wall fraction (pellet) obtained was washed three times with water, subsequently boiled in 50 mM Tris-HCl buffer (pH 7.5) containing 50 mM EDTA, 2% SDS and 40 mM β-mercaptoethanol (β-ME) for 15 min, twice. The sediment obtained after centrifugation (3,000×g, 10 min) was washed five times with water and then incubated in 1 M NaOH containing 0.5 M NaBH₄ at 65°C for 1 h, twice. The insoluble pellet obtained upon centrifugation of this alkali treated sample (3,000×g, 10 min, AI-fraction) was washed with water to neutrality, while the supernatant (AS-fraction) was neutralized and dialyzed against water. Both fractions were freeze-dried and stored at −20°C until further use. Hexose composition in the samples were estimated by gas-liquid chromatography using a Perichrom PR2100 Instrument (Perichrom, Saulx-les-Chartreux, France) equipped with flame ionization detector (FID) and fused silica capillary column (30 m×0.32 mm id) filled with BP1, using meso-inositol as the internal standard. Derivatized hexoses (alditol acetates) were obtained after hydrolysis (4N trifluoroacetic acid/8N hydrochloric acid, 100°C, 4 h), reduction and peracetylation. Monosaccharide composition (percent) was calculated from the peak areas with respect to that of the internal standard.

ROS formation was evaluated by means of the probe 2′,7′-dichlorofluorescin-diacetate (H2DCF-DA). H2DCF-DA is a non-fluorescent permeant molecule that passively diffuses into cells, where the acetates are cleaved by intracellular esterases to form H2DCF, and thereby traps it within the cell. In the presence of intracellular ROS, H2DCF is rapidly oxidized to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). After cell treatment, previously reported, cells were collected, washed twice with phosphate buffer saline (PBS), and then incubated in PBS containing H2DCF-DA (10 µM) at 37 °C. After 15 min, fluorescence was evaluated using a fluorescence-activated cell sorting (BD FacsCalibur, Milan, Italy) and elaborated with Cell Quest software, as previously reported [23 (link)].

Molecular weight distributions of lyophilized crude EPS were determined by size exclusion chromatography. In brief, crude EPS powder was suspended in 0.1 M NaNO₃ (0.5 mg/mL) and then filtered through a 0.45 μm pore diameter polyvinylidene fluoride membrane (Millipore Corporation, USA). The average molecular weight (MW) was determined by high-performance molecular exclusion chromatography (HPLC-SEC, Agilent 1,100 Series System, Hewlett-Packard, Germany) associated with a refractive index (IR) detector (Ibarburu et al., 2015 (link)). 50 μL of the samples were injected and eluted at a flow rate of 0.95 mL/min (pressure: 120:130 psi) at room temperature using 0.1 M NaNO₃ as mobile phase. Dextrans (0.5 mg/mL) with a molecular weight between 10³ and 2.10⁶ Da (Sigma-Aldrich, USA) were used as standards.
Once the molecular weight distributions were determined, low and high molecular weight fractions that composed the crude EPS obtained at 20°C were separated. For this purpose, EPS solutions (0.2% w/v) were centrifuged through a Vivaspin™ ultrafiltration spin column 100 KDa MWCO, (Sartorious, Goettingen, Germany) for 20 min at 6000 g, eluting only the low MW fraction. Subsequently, high MW fraction retained in the column was eluted using hot distilled water. The eluted fractions were passed through a Vivaspin column (cut-off 30KDa) in order to separate the middle and low MW fraction of EPS.
Monosaccharide composition of crude EPS and their fractions were determined by gas chromatography as previously described (Notararigo et al., 2013 (link)). Briefly, 1–2 mg of EPS were hydrolyzed in 1 mL of 3 M trifluoroacetic acid (1 h at 120°C). The monosaccharides obtained were converted into alditol acetates by reduction with NaBH₄ and subsequent acetylation. The samples were analyzed by gas chromatography in an Agilent 7890A coupled to a 5975C mass detector, using an HP5-MS column with helium as carrier gas at a flow rate of 1 mL/min. For each run, 1 μL of sample was injected (with a Split 1:50) and the following temperature program was performed: the oven was heat to 175°C for 1 min; the temperature was increased to 215°C at a rate of 2.5°C/min and then increased to 225°C at 10°C/min, keeping it constant at this temperature for 1.5 min. Monosaccharides were identified by comparison of retention times with standards (arabinose, xylose, rhamnose, galactose, glucose, mannose, glucosamine and galactosamine) analyzed under the same conditions. Calibration curves were also processed for monosaccharide quantification. Myo-inositol was added to each sample as internal standard.

Ethyl 2-[4-oxo-8-(R-phenyl)-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl]acetates (1–6) belonging to fused azaisocytosine congeners have been synthesised for the purposes of thermal studies according to efficient synthetic approaches previously patented and published [2 ,3 (link)]. The structures of molecules 1–6 have been confirmed by ¹H-NMR/¹³C-NMR spectra and elemental analysis, and established on the basis of the performed ¹³C, ¹H HMBC and HMQC correlations for the ethyl ester of 2-(4-oxo-8-phenyl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)acetic acid (1) [3 (link)]. The purity and homogeneity of all the compounds intended for thermal studies (1–6) have been previously evaluated under reaction and the purification conditions employed. All these ones have been obtained and described [3 (link)] as homogenous, pure, crystalline solids with sharp melting points and microanalyses within ±0.4 percent of the calculated values. They have been reported to reveal not only enhanced anticancer effects in malignant human multiple myeloma cells (MM1R, MM1S) but also antiproliferative activities against human tumours of the breast (T47D) and cervix (HeLa) [3 (link)]. In addition, their mode of anticancer action and very low toxicities towards normal human skin fibroblasts have been previously documented [3 (link)].

In order to test the viability of drug-resistant Colo 320 cells before and after treatments with chemotherapy drugs or synthetic steroids, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays were conducted (first described by Mosmann [42 (link)]). First the toxicity of chemotherapy drugs was determined. Colo 320 cells were seeded in 96-well plates at 10⁴ cells/well density and were left to grow for 24 h. To obtain dose-response curves, cells were treated with a serial dilution of each drug (Bleomycin, Carmustine, Cisplatin, Doxorubicin, Epirubicin; treatment was applied in 200 μL media) for another 24 h (for the applied concentrations, please refer to Supplementary Material Table S1). Then, the media were discarded and replaced with 100 μL fresh media containing 0.5 mg/mL MTT reagent (Sigma-Aldrich, St. Louis, MO, USA). After a 1 h incubation, this media was replaced by 100 μL DMSO to solubilize formazan crystals. Absorbance of the samples was measured at 570 and 630 nm with a Synergy HTX microplate reader (BIOTEK^®, Santa Clara, CA, USA). Untreated cells were considered as 100% cell viability during data analysis. IC₅₀ and the Hill-slope values were obtained from the measured data. The IC₁₀–IC₉₀ values for each drug were calculated from the IC₅₀ and the Hill-slope values.
We tested also the toxicity of androstano-arylpyrimidine 17-acetates. For this, drug-sensitive Colo 205 and multidrug-resistant Colo 320 cells were exposed to compounds 10a, 10d, 10e, 10f, and 10g in a fixed 20 μM concentration for 24 h, then MTT viability assay was performed. Cell seeding and absorbance measurement were carried out in the same way as described above.
For the combinational treatments (chemotherapy drug + steroid), each chemotherapy drug was applied in its previously determined respective IC₃₀–IC₇₀ concentration (in the case of each drug, the appropriate concentrations are presented in Supplementary Material Table S2, indicated in the first column of the table next to the drug name) together with each androstano-arylpyrimidine 17-acetate (10a, 10d, 10e, 10f, and 10g). The steroids were used in a fixed 20 μM concentration. Based on these treatments, certain combinations were selected to be utilized later for an apoptosis detection assay.